DESCRIPTION ( applicant's abstract): The studies proposed in this new grant application will examine the structure and mechanism of action of the enzyme phosphonatase and extend structural and mechanistic studies to other members of the haloacid dehalogenase (HAD) enzyme superfamily. Each enzyme of this superfamily uses a conserved Asp residue to form either an acylphosphate-enzyme intermediate or an alkyl ester-enzyme intermediate. This chemistry is supported by a common structural scaffold. Phosphonatase catalyzes the hydrolysis of phosphonoacetaldehyde (P-Ald) to acetaldehyde and orthophosphate. In conjunction with 2-aminoethylphosphonate transaminase, phosphonatase functions in a two-step biodegradative pathway used to recycle P, N, and C from the ubiquitous natural phosphonate, 2-aminoethylphosphonate. Despite the wide range of known biological activities associated with natural and synthetic phosphonates, the enzymology of phosphonate metabolism is poorly characterized. The goal of these studies is to derive an understanding of the process of enzyme catalyzed C-P bond cleavage using phosphonatase as the model system. The first set of experiments proposed will test mechanistic models based on the recently determined phosphonatase X-ray structure (Allen laboratory) and on previous mechanistic studies (Dunaway-Mariano laboratory). Site-directed mutagenesis coupled with transient kinetic analysis will be used to test the chemical steps of the models. Crystallographic structure determinations carried out on dead-end complexes formed using substrate analogues, enzyme mutants, and chemically modified enzymes will be used to capture the structures of proposed reaction intermediates. The second set of experiments proposed will examine the active-site diversification of the HAD enzyme superfamily. The ability of phosphonatase to catalyze the reactions of other family members will be determined and protein engineering will be used to swap catalytic activities of two family members. To further probe the catalytic plasticity of the superfamily, the structure and mechanism of beta-phosphoglucomutase, a phosphotransferase from the HAD family will be examined. The goal of these studies is to derive an understanding of how the enzyme superfamily active site has been adapted to catalyze C-X, P-O and C-P bond cleavage in a variety of different substrate structures.